This application relates to a new method for sequencing nucleic acids without the use of electrophoresis, radioactivity, or fluorescense. The technique analyzes the production of inorganic pyrophosphate from nucleic acid polymerization reactions, and has the advantages of being simple, cost effective, and completely automatable.
Current methods used to sequence nucleic acids all involve electrophoresis of single stranded nucleic acid fragments (ssDNA or ssRNA) generated by either chain terminating nucleotides in the Sanger dideoxy sequencing technique, selective enzymatic fragmentation of strands of RNA or selective chemical degradation of DNA by the Maxam and Gilbert technique. All of these methods involve separation of nucleic acid fragments in polyacrylamide gels and measurement of their location by radioactivity. New DNA sequencing machines recently made commercially available use fluorescense instead of radioactivity to detect the ssDNA fragments. However, these newer techniques involve expensive equipment, specialized chemicals, and still require intensive labor and careful technique to carry out the procedure successfully.
The present invention takes a completely different approach to the problem of sequencing nucleic acids based on the precise measurement of the inorganic pyrophosphate (PPi) generated during nucleic acid polymerization reactions, such as the polymerization of deoxynucleotide triphosphates (dNTP) with a ssDNA template-primer complex catalyzed by DNA polymerase shown below: EQU ssDNA-primer+dNTP.fwdarw.ssDNA-(primer+dNMP)+PPi
As shown, one PPi is generated for each dNTP consumed and incorporated into the DNA. If, for example x moles of ssDNA template-primer all have their primer chains extended by one base, then x moles of PPi will be generated. If the primer chains are extended by two bases, then 2x moles of PPi will be generated, etc. Thus, by precisely measuring PPi, it is possible to determine whether or not a polymerization reaction has occurred, and if so, determine how many nucleotides have been incorporated in the growing primer chain.
Because PPi is formed in a number of biosynthetic pathways, a number of different methods have been developed to assay for PPi. One such assay uses two enzymes, ATP-sulfurylase and firefly luciferase, to produce a light emission proportional in intensity to the amount of PPi. Nyren et al., 151 Analytical Biochemistry 504 (1985). The reactions occurring in this assay are ##STR1##
The second half of this reaction scheme has also been used as an assay for ATP. Luciferase, however, is not entirely specific for ATP as a high energy substrate and can also react with deoxyadenosine-5'-triphosphate (dATP) and to a lesser extent may react with other nucleoside triphosphates as well. Moyer et al., 131 Analytical Biochemistry 187 (1983).
The measurement of PPi has not previously been considered as a means of determining a nucleic acid sequence. As will be shown hereinbelow, however, this approach provides a simple method for rapid determination of nucleic acid sequences.